Immune modulation is a critical aspect of the treatment of a number of diseases and disorders. T cells in particular play a vital role in fighting infections and have the capability to recognize and destroy cancer cells. Enhancing T cell mediated responses is a key component to enhancing responses to therapeutic agents. However, it is critical in immune modulation that any enhancement of an immune response is balanced against the need to prevent autoimmunity as well as chronic inflammation. Chronic inflammation and self-recognition by T cells is a major cause for the pathogenesis of systemic disorders such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus. Furthermore, long term immunosuppression is required in preventing rejection of transplanted organs or grafts.
The mechanisms that prevent T-cell mediated autoimmune reactions are collectively known as T cell “tolerance”. Tolerance can occur by removing antigen specific T cells from the population, which occurs both in the thymus and the periphery. In addition, tolerance can be maintained by ‘turning off’ certain antigen specific T cells or rendering them anergic. When T cells recognize an antigen under conditions that promote anergy, these same cells later fail to respond to antigen upon rechallenge even under normally activating conditions. Anergy is induced when T cell receptor engagement (Signal 1) occurs in the absence of co-stimulation (Signal 2). A major set of co-regulatory molecules is in the B7-CD28 family.
In addition to anergy and deletion, recently it has become clear that regulatory T cells play an important role in maintaining tolerance. Regulatory T cells suppress auto-reactive T cells. Thus, as the level of regulatory T cells decreases, the potential for autoimmunity rises. Interestingly, tumors have been shown to evade immune destruction by impeding T cell activation through inhibition of co-stimulatory factors in the B7-CD28 and TNF families, as well as by attracting regulatory T cells, which inhibit anti-tumor T cell responses (see Wang (2006) Immune Suppression by Tumor Specific CD4+ Regulatory T cells in Cancer. Semin. Cancer. Biol. 16:73-79; Greenwald, et al. (2005) The B7 Family Revisited. Ann. Rev. Immunol. 23:515-48; Watts (2005) TNF/TNFR Family Members in Co-stimulation of T Cell Responses Ann. Rev. Immunol. 23:23-68; Sadum, et al. (2007) Immune Signatures of Murine and Human Cancers Reveal Unique Mechanisms of Tumor Escape and New Targets for Cancer Immunotherapy. Clin. Canc. Res. 13(13): 4016-4025).
Autoimmune diseases develop when the body's immune system fails to recognize normal body tissues and attacks and destroys them as if they were foreign rather than attacking an outside organism. There are nearly 150 autoimmune disorders with no currently known cures. Although the cause is not fully understood, pioneering work by Rose, Witebsky, Roitt and Doniach provided evidence that autoimmune diseases result at least in part from loss of T cell tolerance. An essential prerequisite for the pathogenesis of autoimmune diseases is indeed the breakage of immunological tolerance, which leads to the immune system mounting an effective and specific immune response against self determinants. Several theories exist as to what causes this breakdown, including the breakdown of “clonal deletion theory”, according to which self-reactive lymphoid cells are destroyed during the development of the immune system in an individual, the breakdown of “clonal anergy theory”, in which self-reactive T- or B-cells become inactivated in the normal individual and cannot amplify the immune response, the breakdown of “idiotype network theory”, wherein a network of antibodies capable of neutralizing self-reactive antibodies exists naturally within the body, and the “suppressor population theory”, wherein regulatory T-lymphocytes prevent or limit autoaggressive immune responses.
Adenosine modulates diverse physiological functions including induction of sedation, vasodilatation, suppression of cardiac rate and contractility, inhibition of platelet aggregation, stimulation of gluconeogenesis and inhibition of lipolysis (see, Stiles (1986) Trends Pharmacol. Sci. 7:486; Williams, (1987) Ann. Rev. Pharmacol. Toxicol. 27:315; Rarnkumar et al., (1988) Prog. Drug. Res. 32:195). In addition, adenosine and some adenosine analogs that non-selectively activate adenosine receptor subtypes decrease neutrophil production of inflammatory oxidative products (Cronstein et al., (1986) Ann. N.Y. Acad. Sci. 451:291; Roberts et al., (1985) Biochem. J., 227:669; Schrier et al., (1986) J. Immunol. 137:3284; Cronstein et al., (1987) Clinical lmmunol. Immunopath. 42:76).
Adenosine binds to P1 purinergic receptors, which are members of the G protein-coupled receptor family. Four subtypes of adenosine receptors have been cloned: A1, A2a, A2B, and A3. The four subtypes have the hallmark structural characteristics that are common to G protein-coupled receptors, including seven putative transmembrane spanning domains, an extracellular NH2 terminus, cytoplasmic COOH terminus, and a third intracellular loop that is important in binding G proteins.
The A2a receptor cDNA, which has been cloned from several species including humans, encodes a protein of 45 kDa, larger than the molecular masses of the other subtypes. This is primarily due to the additional 80-90 amino acids of the COOH-terminal tail. The overall amino acid identity is 90% among species, with most of the differences occurring in the second extracellular loop and the long COOH-terminal domain. The COOH-terminal domain has several serine and threonine residues that are potential phosphorylation sites. A2a adenosine receptors stimulate adenylyl cyclase and increase the production of cAMP by coupling to stimulatory G proteins (Gs) or to Golf in certain tissues. In addition to the cAMP-protein kinase A (PKA) pathway, recent studies indicate that serine/threonine protein phosphatase, mitogen-activated protein kinase (MAP kinase), PKC, and phospholipase D may participate in mediating the effects of A2a adenosine receptor activation. Further, the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs, also known as Epac1 and Epac2) may also participate in mediating the effects of these receptors.
Studies have indicated that adenosine has a direct effect on hematopoietic and endothelial cells to reduce inflammation (for a review, see Linden (2001) Molecular approach to adenosine receptors: receptor mediated mechanisms of tissue protection. Annu Rev Pharmacol Toxicol 41: 775-787). Evidence for an anti-inflammatory role of A2a adenosine receptor activation comes from a variety of studies both in vivo and in vitro (Cronstein et al. (1990) The adenosine/neutrophil paradox resolved: human neutrophils possess both A1 and A2 receptors that promote chemotaxis and inhibit O2− generation, respectively. J Clin Invest 85: 1150-1157; Schrier and Imre (1986) The effects of adenosine agonists on human neutrophil function (Abstract). J Immunol 137: 3284; Sullivan et al. (1995) The specific type IV phosphodiesterase inhibitor rolipram combined with adenosine reduces tumor necrosis factor-a (TNF-a)-primed neutrophil oxidative activity. Int J Immunopharmacol 17: 793-803). This physiological role of endogenous adenosine became apparent after the demonstration that activated neutrophils or endothelial cells release and respond to adenosine. Monocytes accumulate more slowly at sites of inflammation than neutrophils and contribute to the inflammatory process by producing and releasing cytokines. The results of several studies indicate that the proinflammatory cytokine TNF-α, is regulated by A2a adenosine receptors (Bouma et al. (1994) Differential regulatory effects of adenosine on cytokine release by activated human monocytes. J Immunol 153:4159-4168; Eigler et al. (1997) Endogenous adenosine curtails lipopolysaccharide-stimulated tumour necrosis factor synthesis. Scand J Immunol 45: 132-139; Hasko et al. (1996) Adenosine receptor agonists differentially regulate IL-10, TNF-a, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J Immunol 96: 4634-4640; Reinstein et al. (1994) Suppression of lipopolysaccharidestimulated release of tumor necrosis factor by adenosine: evidence for A2 receptors on rat Kupffer cells. Hepatology 19: 1445-1452).
On the basis of the evidence that activation of A2a adenosine receptors regulates factors that attenuate inflammation, studies have been performed using selective A2a agonists in tissue to determine whether activation of A2a receptors confers tissue protection. In many of these studies, the observation that A2a agonist-induced tissue protection was associated with a reduction of factors associated with inflammation suggested that A2a agonists contribute to tissue protection by attenuating inflammation, although a direct causal relationship between tissue protection and attenuation of inflammation by A2a agonists has not been proven.
Evidence has accumulated that adenosine accumulation in hypoxic conditions can lead to activation of A2a receptors and, in certain instances, can cause inhibition of immune cells, in particular, of T lymphocytes.
Ohta and Sitkovsky have proposed that adenosine, when acting on A2a receptors, protects tissues from excessive inflammation (Ohta, and Sitkovsky (2001) Role of G-protein-coupled adenosine receptors in down-regulation of inflammation and protection from tissue damage. Nature 414(6866):916-20). Using an A2a receptor knock-out mouse, Ohta et al. showed that, while sub-threshold doses of an inflammatory stimulus caused minimal tissue damage in wild-type mice, such doses were sufficient to induce extensive tissue damage, more prolonged and higher levels of pro-inflammatory cytokines, and death of animals deficient in the A2a adenosine receptor. Additional observations were made in studies of model systems of inflammation and liver damage as well as during bacterial endotoxin-induced septic shock.
Kinsel and Sitkovsky overviewed possible targeting of certain G protein coupled receptors, including A2a receptors, in manipulating inflammation in vivo with ligands (Kinsel J F, Sitkovsky M V. (2003) Possible targeting of G protein coupled receptors to manipulate inflammation in vivo using synthetic and natural ligands. Ann Rheum Dis. 62 Suppl 2:ii22-4). The authors state that targeting of these receptors by selective agonists may lead to better protocols of anti-inflammatory treatments, and that inhibiting the Gs protein coupled mediated signaling with antagonists could be explored in studies of approaches to enhance inflammation and tissue damage.
Ohta, et al. have also proposed that the A2a adenosine receptor protects tumors from anti-tumor T cells (Ohta, et al. (2006) A2a adenosine receptor protects tumors from antitumor T cells. PNAS 103(35):13132-7). Again using A2a receptor deficient mice, the investigators showed that approximately 60% of tumor cells were rejected when compared to no rejection in normal mice. The investigators also showed that treatment using an A2a receptor antagonist improved inhibition of tumor growth, destruction of metastases and prevention of neovascularization by anti-tumor T cells. In all cases, the treatment was continuous during the timeframe, with no suggestion of long term effects.
PCT Publication No. WO 03/050241 by Sitkovsky and Ohta describes the methods to increase an immune response to an antigen, increasing vaccine efficacy or increasing an immune response to a tumor antigen or immune cell-mediated tumor destruction by administering an agent that inhibits extracellular adenosine or inhibits adenosine receptors.
Sullivan described the role of endogenous adenosine in blocking potentially destructive inflammatory cascades by binding to A2a adenosine receptors and decreasing activation of platelets, leukocytes and endothelial cells (Sullivan G W. (2003) Adenosine A2a receptor agonists as anti-inflammatory agents. Curr Opin Investig Drugs. 4(11):1313-9). Sullivan also reviews potential disease targets for A2a receptor agonist treatment, including in allergen-induced inflammation, ischemia-reperfusion injury, sepsis and autoimmune diseases.
Kinsel and Sitkovsky overviewed possible targeting of certain G protein coupled receptors, including A2a receptors, in manipulating inflammation in vivo with ligands (Kinsel J F, Sitkovsky M V. (2003) Possible targeting of G protein coupled receptors to manipulate inflammation in vivo using synthetic and natural ligands. Ann Rheum Dis. 62 Suppl 2:ii22-4). The authors state that targeting of these receptors by selective agonists may lead to better protocols of anti-inflammatory treatments.
Ulusal et al. conducted in vivo experimental studies to investigate whether A2a receptor agonists reduce allostimulatory functions of dendritic cells, for example through modulation of surface expression of the costimulatory molecules and down-regulation of cytokines (Ulusal B G, et al. (2006) The effect of A2a adenosine receptor agonist on composite tissue allotransplant survival: an in vivo preliminary study. J Surg Res. 131(2):261-6). The authors state that the results from this study showed that A2a adenosine receptor agonist treatment does not prolong composite tissue allograft survival.
Sevigny, et al. investigated the in vitro and in vivo effect of A2a receptor agonists to attenuate allogenic immune activation (Sevigny C P, et al. (2007 Apr. 1) Activation of adenosine2a receptors attenuates allograft rejection and alloantigen recognition. J Immunol 178(7):4240-9). The authors state that the results indicated that A2a receptor agonists attenuate allogenic recognition by action on both T lymphocytes and APCs in vitro and delayed acute rejection in vivo and may represent a new class of compounds for induction therapy in organ transplantation.
Nemeth, et al. investigated adenosine receptor activation in type I diabetes and suggest that adenosine receptor ligands could be potential candidates for treatment of type I diabetes and could be promising targets in autoimmune disease (Németh Z H, et al. (2007) Adenosine receptor activation ameliorates type 1 diabetes. FASEB J. epub).
There remains a need for therapies that provide long term enhancement of immune responses to specific antigens, particularly for treatment and prevention of abnormal cell proliferation and for treatment of infectious diseases and disorders. There also remains a need for treatments that provide long term, targeted immune suppression and reduce the need for standard immunosuppressive therapies in certain disorders, in particular in the area of transplantation and autoimmunity.
It is an object of the present invention to provide methods of treatment that allow simplified treatment protocols and enhance immune responses against certain antigens. It is a specific object of the invention to provide improved methods of preventing or treating abnormal cell proliferation and infectious diseases in a host. It is a separate object of the present invention to provide more effective therapeutic regimes to reduce the need for long term treatment with immunosuppressive therapies in a host.